The invention relates to the field of genetic pharmacology and in particular to the screening of compounds for potential therapeutic activity using nematodes, principally but not exclusively, the nematode Caenorhabditis elegans. Specifically, the invention relates to the use of nematodes modified to have characteristics suitable for compound screening and to methods of selecting suitably modified nematodes from a population of nematodes. Caenorhabditis elegans is a nematode worm which occurs naturally in the soil but can be grown easily in the laboratory on nutrient agar or in liquid nutrient broth inoculated with bacteria, preferably E. coli, on which it feeds. Each worm grows from an embryo to an adult worm of about 1 mm long in three days or so. As it is fully transparent at all stages in its life, cell divisions, migrations and differentiation can be seen in live animals. Furthermore, although its anatomy is simple its somatic cells represent most major differentiated tissue types including muscles, neurons, intestine and epidermis. Accordingly, differences in phenotype which represent a departure from that of a wild-type worm are relatively easily observed, either directly by microscopy or by using selective staining procedures. Many C. elegans mutants have been identified and their phenotype described, for example see C. elegans II Ed. Riddle, Blumenthal, Meyer and Priess, Cold Spring Harbor Laboratory Press, 1997. Furthermore, standard methods are known for creating mutant worms with mutations in selected C. elegans genes, for example see J. Sutton and J. Hodgkin in “The Nematode Caenorhabditis elegans”, Ed. by William B. Wood and the Community of C. elegans Researchers CSHL, 1988 594–595; Zwaal et al, “Target-Selected Gene Inactivation in Caenorhabditis elegans by using a Frozen Transposon Insertion Mutant Bank” 1993, Proc. Natl. Acad. Sci. USA 90 pp 7431–7435; Fire et al, Potent and Specific Genetic Interference by Double-Stranded RNA in C. elegans 1998, Nature 391, 860–811.
The C. elegans genome is now entirely sequenced and is available in a public database of the Wellcome Trust Sanger Institute. As a result of the C. elegans genome sequencing project, it has emerged that C. elegans comprises genes which have equivalents that are widely distributed in most or all animals including humans.
The possibility that C. elegans might be useful for establishing interactions between external molecules and specific genes by comparison of C. elegans phenotypes which are generated by exposure to particular compounds and by selected mutations is considered by Rand and Johnson in Methods of Cell Biology, Chapter 8, volume 84, Caenorhabditis elegans: Modern Biological analysis of an Organism Ed. Epstein and Shakes, Academic Press, 1995 and J. Ahringer in Curr. Op. in Gen. and Dev. 7, 1997, 410–415.
Rand and Johnson in particular describe compound screening assays in which varying concentrations of the compound to be tested are added to nutrient agar or broth which is subsequently seeded with bacteria and then inoculated with worms. Any phenotypic changes in the worm as a result of exposure to the compound are then observed.
Although the nematode and in particular, C. elegans, is proving a powerful and efficient tool in the identification or discovery of pharmacologically active molecules, the presently known techniques for compound screening are not without drawbacks. A particular problem is that C. elegans, like higher organisms, is endowed with physiological barriers which prevent or minimise the ingress of foreign and potentially harmful substances. Because it normally lives in the dirt, this nematode has evolved a high number of pgp and mdr genes (Table 2) and hence has a high detoxification capacity which is a disadvantages for laboratory drug discovery purposes. In the past the only way in which these barriers have been overcome is to expose the worm to a high concentration of the compound to be tested, for example in the millimolar range. This is inconvenient because many of the compounds which might be screened in C. elegans are either only available in micromolar quantities or it is not economically feasible to use concentrations any higher than this. Furthermore, high concentrations of compounds may lead to death of the bacteria and hence starvation of the nematodes. It has also been observed that compounds crystallize when applied in high concentration.
If a compound is to reach a particular target in C. elegans it must overcome barriers in the pharynx, and in the gut and/or the cuticle. C. elegans feeds by taking in liquid containing its food (e.g. bacteria). It then spits out the liquid, crushes the food particles and internalises them into the gut lumen. This process is performed by the muscles of the pharynx. The process of taking up liquid and subsequently spitting it out is called pharyngeal pumping. Since this pumping process is stimulated to take place mainly in the presence of food, the pharynx is not pumping all the time in wild-type C. elegans. As a result, if worms are placed in a liquid culture in the presence of a dye a slow uptake and large variance in uptake amongst individual worms is observed.
Once a test compound has been internalised then the gut itself is a further frontier that may prevent the test compounds reaching their target site in the worm. This may be due to the presence of modifying and detoxifying enzymes in the gut, examples of which are the multi-drug resistance proteins (P-glycoproteins) and the multi-drug resistance related proteins.
Finally, C. elegans possesses a natural barrier to external substances which is the cuticle. The cuticle covers the outermost surfaces of the worm and also lines the pharynx and the rectum. It comprises mainly collagens and is substantially impermeable.